The invention relates to an assay chip for investigation of the functionality of membrane proteins and their interactions with molecules. Further, the invention relates to a process for analyzing the functionality of non-lipid molecules, such as a protein, being integrated in a fluid biological effective layer. Furthermore, the invention relates to the use of said assay chip.
Detailed knowledge about protein structures and their related functions is a key to understand molecular processes of life. Due to the powerful molecular cloning and gene expression technologies proteins for the purpose of analytical investigations can be produced in sufficient amounts. Proteomics is presently a very active field in life sciences research. The final aim is to achieve a comprehensive understanding of structure and function of proteins. This knowledge is a prerequisite for a rational design of new drugs. G-protein coupled receptors (GPCR) constitute the largest subgroup of cell membrane receptors and about half of them are considered to be targets for drugs. As for an example, the actual structure of bovine rhodopsin, a model for GPCR proteins, could recently be determined at a 2.8 Å resolution. GPCRs have a glycosylated N-terminal ligand binding site, seven trans-membrane helices and an intracellular G-protein binding domain. Ligand binding on the extracelluar part induces conformational changes in the trans-membrane helices bundle of the receptor protein resulting in a dissociation of the hetero-trimeric G-protein which is bound to the intracellular part of the GPCR into a α- and a βγ-subunit. G-protein are anchored covalently to the lipid bilayer and are currently classified in four families according to the nature of the α-subunits which interact with different target membrane proteins like enzymes or ion channels. After the dissociation of the G-protein the α-subunit laterally diffuse within the lipid bilayer and bind to the target protein and activate it.
Therefore, the composition and the fluidity of the lipid bilayer are in any type of functional analysis and screening process critical issues, especially of high interest when considering an economic process for screening purposes.
Arrays of immobilized GPCRs in micro-spots have recently been used to investigate compounds which specifically bind to this membrane protein. Such high throughput technologies allow a screening across or within receptor families and may be suitable for ligand fishing for orphan GPCRs. The biological response of the current about 140 orphan GPCR receptors are unknown which make it difficult to identity ligands as potentially useful drugs.
Deorphanization of non-olefactory GPCRs is currently a focus in the pharmaceutical industry and recently receptors with potential functions related to cancer and diabetes has been identified. The success in finding new binding compounds depends strongly on full functionality of the target protein as it is the case in the living cell. Allosteric sites on GPCRs which do not overlap with the binding sites for the natural agonist have a number of theoretical advantages over agonist binding sites for drugs such as saturability and high tissue specificity. Thus, allosteric sites are attractive targets sites for drugs which modulate receptor functions by increase or partially decreasing its activity. The binding of allosteric ligand or modulating proteins can result in membrane protein complexes consisting of several components. Again, for the investigation of such interactions highly sensitive functional assay systems are required in which the target membrane protein has to be fully mobile within the lipid bilayer membrane and well accessible.
In almost all cases of the in vitro assay systems as outlined below, biomembranes of unknown composition are immobilized on solid supports which often leads to a restricted fluidity of the lipid bilayer and a structural disturbance of the membrane protein. For that reason, these circumstances are greatly undesired in broad assay processing campagnes since for the drug screening processing the native functions of the proteins are potentially highly disturbed.
A second important class of membrane proteins are ion channels. The voltage-gated sodium channels of eukaryotes are complex membrane proteins composed of more than 2000 amino acids residues. The bacterial sodium channel is much simpler consisting of one-domain of 34 kD and it can be expressed at high rates and is therefore useful for assay technology development. High throughput screening assays use lipid bilayer membranes associated sensitive reporter molecules or fusion constructs consisting of the green fluorescence protein and the ion channel. By the use of the proposed highly sophisticated assay systems, a deeper insight into the function of membrane proteins of interest will be achieved. Both, academic research and drug discovery will profit from this knowledge.
Cell-based assays are well suited to monitor the biological response of GPCRs in their natural environment and are widely used to identify lead compounds in the drug discovery process. In order to understand molecular mechanisms, in vitro tests are required where the number of involved components is reduced. The functionality of membrane proteins in vivo depends on many factors: the composition of the lipid bilayer membrane, biological activation reactions such as phosphorylation or the intracellular Ca++ concentration. The development of robust, functional biomimetic in vitro tests for membrane proteins is a demanding, highly interdisciplinary field. The first step is the reconstitution of the purified expressed recombinant proteins into lipid bilayer membranes, in order to bring them in the functional form. Vesicles consisting of lipid bilayers are commonly used to keep membrane proteins active in buffer solutions. Binding of ligands to proteins present in vesicles has been monitored when they are bound on sensor surfaces using surface plasmon resonance as the detection method. However, such vesicle preparations may require too high quantities of the protein to achieve a sufficiently high sensitivity in the measurements and they are not well suited to investigate many functional aspects. Therefore, several types of hybrid systems have been developed where planar lipid bilayer membranes are immobilized on solid supports.
Free standing membranes which span a hole in a Teflon support appear black. Such black lipid bilayer membranes are difficult to prepare and are extremely fragile since the lipid bilayer of 4 nm thickness has to span holes in the range of 200 μm to 1 mm. For research experiments, this limitation may be acceptable, however, for practical applications in the drug discovery stability of the assay system is a major issue. Therefore, since about 20 years techniques have been developed using supports in order to achieve a higher stability. Membrane proteins have been immobilized by fusion of lipid vesicles to supported lipid bilayers or directly to SiO2. Planar bilayer formation from vesicle adsorbed to hydrophilic surfaces like mica could be visualized.
The changed physical properties of lipid bilayers when supported, however, strongly affect the function of the embedded membrane protein. In order to overcome these problems, lipid bilayer membranes have been immobilized on surfaces via tethers. The resulting cleft between the lipid bilayer membrane and the solid support mimics the intracellular space and the desired functionality of trans-membrane proteins is partially retained. Ligand binding to membrane proteins in the lipid bilayer has been monitored using fluorescence detection. Impedance was measured to determine the function of supported ion channels as well. The conductance of supported lipid bilayer membranes containing an iono-phore changes upon binding of ligands. This effect has been utilized to develop membrane protein based biosensors.
The advantages of a hybrid system are a gain in stability and the feasible preparation procedure. The formation of supported planar lipid bilayer membranes by fusion of vesicles with supported phospholipid monolayers has been established. For optical detection of ligand binding lipid bilayer membranes have been prepared on alkylated carboxylated dextrane polymer which has been immobilized on sensor surfaces in a high density. In a study using micropatterned surfaces it has been shown that hydrophilic-hydrophobic edges promote vesicle fusion. Furthermore, the fusogenic effects of short-chained alcohols, Ca++-ions and PEG are known in the art. These studies show that vesicle composition, fusogenic agents, hydrophilicity, topographic and chemical properties of the surface and temperature are the most important factors influencing the formation of lipid bilayer membranes from vesicles adsorbed to solid supports.
Supported tethered lipid bilayer membranes as achieved according to the methods described above have also some major disadvantages. The composition of the fluid in the reservoir between the lipid bilayer membrane and the support can hardly be controlled and the fluidity of the immobilized lipid bilayer membrane is restricted. This constitutes a tremendous drawback since the functionality of membrane proteins is related to the fluidity of the lipid bilayer and their mobility therein. Additionally, the incorporation of membrane proteins with large trans-membrane loops—the acetylcholine receptor for instance has a 5 nm large intracellular part—are impeded since the intracellular space between the lipid bilayer and the support gained by a tether are too small for larger trans-membrane loops.
Furthermore, only one side of the tethered lipid bilayer membrane is accessible which makes it difficult to explore trans-membrane changes or transport of molecules. The non-native environment may result in an impairment of the functionality and in screenings physiological receptor functions may not be detected.